Over the past decade vast improvement in disease-free survival of poor prognosis patients with hematologic malignancies and certain solid tumors, such as breast cancer, has been achieved. In large part this has been due to treatment with high-dose chemotherapy regimens followed by hematopoietic progenitor cell support. As new technologies emerge for the isolation and ex vivo expansion of progenitor cell fractions for transplantation into these patients, the effect that these manipulations have on rare stem cell populations is largely unknown. Currently assays do not exist for measuring human repopulating stem cells. The goal of this project is to use the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model to identify and characterize the most primitive precursors that represent the earliest stages of human hematopoiesis. Results from these studies will provide information regarding the developmental potential of the most primitive elements of the human hematopoietic system and ultimately provide methods for assaying the effects that in vitro manipulations may have on that potential. The first aim of this work is to define the characteristics of long-term hematopoietic precursors that are capable of repopulating NOD/SCID mice. An assay based on multilineage repopulation of secondary recipients will be developed to characterize the long-term repopulating potential of human precursors, fractionated on the basis of physical or functional criteria. Following engraftment with human progenitor cell fractions, it will be determined if the number of long-term cells can be manipulated in vivo with various growth factors. A second aim is to assess the effects that in vitro manipulations have on the long-term potential of progenitor cell populations previously identified. Here growth factor combinations that induce signaling through gp130 and/or tyrosine kinase receptors, as well as the presence of stromal cell layers, will be assessed in order to find conditions that optimally preserve long-term repopulation. The final goal of these studies is to correlate patient engraftment with repopulation in the mouse model using the same cell fraction subsequent to ex vivo expansion under clinical conditions. If such a correlation can be demonstrated, it will suggest that the mouse is detecting the cell responsible for human engraftment and enhance the validity of using the mouse model for predicting the long-term repopulation potential of human hematopoietic cell fractions. This result will greatly facilitate the translation to the clinic of results from the other portions of this proposal. The outcome of these studies will provide us with a better understanding of the human precursors responsible for long-term maintenance of the hematopoietic system and may provide methods for the enhancement of that activity. This will ultimately lead to improved therapies for cancer patients as well as for patients with a number of blood cell disorders.